RESUMO
Background: Cutaneous lupus erythematosus (CLE) is an interferon (IFN) -driven autoimmune skin disease characterized by an extensive cytotoxic lesional inflammation with activation of different innate immune pathways. Aim of our study was to investigate the specific role of Janus kinase 1 (JAK1) activation in this disease and the potential benefit of selective JAK1 inhibitors as targeted therapy in a preclinical CLE model. Methods: Lesional skin of patients with different CLE subtypes and healthy controls (N = 31) were investigated on JAK1 activation and expression of IFN-associated mediators via immunohistochemistry and gene expression analyses. The functional role of JAK1 and efficacy of inhibition was evaluated in vitro using cultured keratinocytes stimulated with endogenous nucleic acids. Results were confirmed in vivo using an established lupus-prone mouse model. Results: Proinflammatory immune pathways, including JAK/STAT signaling, are significantly upregulated within inflamed CLE skin. Here, lesional keratinocytes and dermal immune cells strongly express activated phospho-JAK1. Selective pharmacological JAK1 inhibition significantly reduces the expression of typical proinflammatory mediators such as CXCL chemokines, BLyS, TRAIL, and AIM2 in CLE in vitro models and also improves skin lesions in lupus-prone TREX1-/- -mice markedly. Conclusion: IFN-associated JAK/STAT activation plays a crucial role in the pathophysiology of CLE. Selective inhibition of JAK1 leads to a decrease of cytokine expression, reduced immune activation, and decline of keratinocyte cell death. Topical treatment with a JAK1-specific inhibitor significantly improves CLE-like skin lesions in a lupus-prone TREX1-/- -mouse model and appears to be a promising therapeutic approach for CLE patients.
Assuntos
Azetidinas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Ácidos Isonicotínicos/uso terapêutico , Janus Quinase 1/antagonistas & inibidores , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Animais , Azetidinas/farmacologia , Linhagem Celular , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Exodesoxirribonucleases/deficiência , Regulação da Expressão Gênica , Humanos , Ácidos Isonicotínicos/farmacologia , Janus Quinase 1/biossíntese , Janus Quinase 1/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Líquen Plano/enzimologia , Lúpus Eritematoso Cutâneo/enzimologia , Lúpus Eritematoso Discoide/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Fosfoproteínas/deficiência , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Aberrant expression of iron(II)- and 2-oxoglutarate-dependent JumonjiC histone demethylases has been linked to cancer. Potent demethylase inhibitors are drug candidates and biochemical tools to elucidate the functional impact of demethylase inhibition. METHODS & RESULTS: Virtual screening identified a novel lead scaffold against JMJD2A with low-micromolar potency in vitro. Analogs were acquired from commercial sources respectively synthesized in feedback with biological testing. Optimized compounds were transformed into cell-permeable prodrugs. A cocrystal x-ray structure revealed the mode of binding of these compounds as competitive to 2-oxoglutarate and confirmed kinetic experiments. Selectivity studies revealed a preference for JMJD2A and JARID1A over JMJD3. CONCLUSION: Virtual screening and rational structural optimization led to a novel scaffold for highly potent and selective JMJD2A inhibitors.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Isonicotínicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Pró-Fármacos/farmacologia , Pirimidinas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Isonicotínicos/síntese química , Ácidos Isonicotínicos/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
In our experimental approach we examined how potato leaves exposed to a chemical agent might induce nitric oxide (NO) dependent biochemical modifications for future mobilization of an effective resistance to Phytophthora infestans. After potato leaf treatment with one of the following SAR inducers, i.e. ß-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA) or Laminarin, we observed enhanced NO generation concomitant with biochemical changes related to a slight superoxide anion (O2(-)) and hydrogen peroxide (H2O2) accumulation dependent on minimal NADPH oxidase and peroxidase activities, respectively. These rather normoergic changes, linked to the NO message, were mediated by the temporary down-regulation of S-nitrosoglutathione reductase (GSNOR). In turn, after challenge inoculation signal amplification promoted potato resistance manifested in the up-regulation of GSNOR activity tuned with the depletion of the SNO pool, which was observed by our team earlier (Floryszak-Wieczorek et al., 2012). Moreover, hyperergic defense responses related to an early and rapid O2(-)and H2O2 overproduction together with a temporary increase in NADPH oxidase and peroxidase activities were noted. BABA treatment was the most effective against P. infestans resulting in the enhanced activity of ß-1,3-glucanase and callose deposition. Our results indicate that NO-mediated biochemical modifications might play an important role in creating more potent defense responses of potato to a subsequent P. infestans attack.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Óxido Nítrico/farmacologia , Phytophthora infestans/parasitologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Solanum tuberosum/efeitos dos fármacos , Aldeído Oxirredutases/efeitos dos fármacos , Aldeído Oxirredutases/metabolismo , Aminobutiratos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucanos/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácidos Isonicotínicos/farmacologia , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Doenças das Plantas/parasitologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Proteínas de Plantas/efeitos dos fármacos , Polissacarídeos/farmacologia , Solanum tuberosum/imunologia , Solanum tuberosum/parasitologia , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
We investigated how potato exposed to a chemical agent could activate nitric oxide (NO)-dependent events facilitating more potent defense responses to a subsequent pathogen attack. Obtained data revealed that all applied inducers, i.e., ß-aminobutyric acid (BABA), γ-aminobutyric acid (GABA), laminarin, or 2,6-dichloroisonicotinic acid (INA), were active stimuli in potentiating NO synthesis in the primed potato. It is assumed, for the mechanism proposed in this paper, that priming involves reversible S-nitrosylated protein (S-nitrosothiols [SNO]) storage as one of the short-term stress imprint components, apart from epigenetic changes sensitized by NO. Based on BABA- and GABA-induced events, it should be stated that a rise in NO generation and coding the NO message in SNO storage at a relatively low threshold together with histone H2B upregulation might create short-term imprint activation, facilitating acquisition of a competence to react faster after challenge inoculation. Laminarin elicited strong NO upregulation with an enhanced SNO pool-altered biochemical imprint in the form of less effective local recall, nevertheless being fully protective in distal responses against P. infestans. In turn, INA showed the most intensified NO generation and abundant formation of SNO, both after the inducer treatment and challenge inoculation abolishing potato resistance against the pathogen. Our results indicate, for the first time, that a precise control of synthesized NO in cooperation with reversible SNO storage and epigenetic modifications might play an important role in integrating and coordinating defense potato responses in the priming phenomenon.
Assuntos
Óxido Nítrico/metabolismo , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/metabolismo , Aminobutiratos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucanos , Ácidos Isonicotínicos/farmacologia , Polissacarídeos/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
Forty-one compounds including two new constituents, senecainin A (1) and 3-methoxyisonicotinic acid (2), were characterized from the methanol extracts of the whole plant of Senecio scandens. The structures of the new compounds were comprehensively established with the aid of 1D and 2D NMR spectroscopic and mass spectrometric analyses. The chemical structures of known compounds were identified by comparison of their spectroscopic and physical data with those reported in the literature. In addition, the antioxidant activity of some of the isolates was examined in the DPPH free radical scavenging assay. Among the tested compounds, (-)-monoepoxylignanolide, (-)-pinoresinol and (-)-epi-pinoresinol displayed significant antioxidant bioactivity.
Assuntos
Antioxidantes/farmacologia , Cicloexanonas/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Ácidos Isonicotínicos/farmacologia , Senécio/química , Antioxidantes/isolamento & purificação , Compostos de Bifenilo/química , Cicloexanonas/isolamento & purificação , Medicamentos de Ervas Chinesas/isolamento & purificação , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/química , Ácidos Isonicotínicos/isolamento & purificação , Estrutura Molecular , Picratos/químicaRESUMO
NF-kappaB activation is clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The prominent role of IkappaB kinase-2 (IKK-2) in regulating NF-kappaB signaling in response to proinflammatory stimuli has made IKK-2 a primary anti-inflammation therapeutic target. PHA-408, a potent and selective IKK-2 inhibitor, was identified internally and used for our studies to assess this target. In early in vivo studies, PHA-408 demonstrated efficacy at high doses; however, the correlation between PHA-408 exposure and efficacy could not be established using standard dosing paradigms for the rat disease models. Similar concerns arose from early in vivo safety studies where appropriate NOAEL margins were not achieved. Following a full investigation of the physicochemical properties of the molecule and pharmacokinetic modeling, an oral steady-state delivery strategy was designed to administer PHA-408 to the rat for both efficacy and safety studies. Using this steady-state delivery, a clear dose-response relationship was established between plasma concentrations of PHA-408 and efficacy in the rat arthritis model. The same steady-state delivery approach was used to demonstrate the target safety. In summary, a combination of pharmacokinetic modeling with a steady-state delivery approach allowed us to establish confidence in both the mechanism and safety of the target.
Assuntos
Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Quinase I-kappa B/antagonistas & inibidores , Indazóis/administração & dosagem , Ácidos Isonicotínicos/administração & dosagem , Administração Oral , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Artrite Experimental/tratamento farmacológico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Indazóis/efeitos adversos , Indazóis/farmacocinética , Indazóis/farmacologia , Ácidos Isonicotínicos/efeitos adversos , Ácidos Isonicotínicos/farmacocinética , Ácidos Isonicotínicos/farmacologia , Masculino , Modelos Biológicos , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
In view of a possible application to Fe and Al chelation therapy, 2-methyl-3-hydroxy-4-pyridinecarboxylic acid (DT2) was synthesised, and its complex formation, electrochemical and cytotoxic properties were studied. The complexing properties of DT2 towards Fe(III) and Al(III) were investigated in aqueous 0.6 m (Na)Cl at 25 degrees C by means of potentiometric titrations, UV-vis spectrophotometry, and 1H NMR spectroscopy. DT2 is a triprotic acid (H3L+) having pKa1 = 0.47, pKa2 = 5.64 and pKa3 = 11.18. The metal-ligand complexes observed in solution and their corresponding stability constants (log beta values) are the following: FeLH (19.38), FeL (16.01), FeLH(-1) (12.28), FeL2H2 (37.29), FeL3H3 (53.41), FeL3H2 (47.99), FeL3H (41.21) and FeL3 (34.1); AlLH (17.43), AlL2H2 (33.74), AlL2H (27.6), AlL3H3 (48.72), AlL3H2 (42.67), AlL3H (35.8) and AlL3 (27.92). The complex formation between DT2 and Fe(II) was studied by UV-vis: the weak complex FeLH (log beta = 15.8) was detected. DT2 shows a lower complexation efficiency with Fe(III) and Al(III) than that of other available chelators, but higher than that of its non-methylated analogue 3-hydroxy-4-pyridinecarboxylic acid (DT0). The electrochemical behaviour of DT2 was investigated by means of cyclic voltammetry, indicating that the oxidation of the ligand proceeds through a two electron process with a CECE mechanism. Voltammetric curves suggest that the oxidation or the reduction of DT2 in vivo is unlikely. According to the thermodynamic data, also the Fe(III)-DT2 complexes do not undergo redox cycling at physiological pH. Amperometric titrations of solutions containing Fe(III) and DT2 at pH = 5 indicated the same Fe(III) : ligand stoichiometric ratio as calculated from potentiometric data. The toxicity of DT2 and of other simple hydroxypyridinecarboxylic acids was investigated in vitro and no cytotoxic activity was observed (IC50 > 0.1 mM) on cancer cell lines and also on primary human cells, following a three day exposure.
Assuntos
Alumínio/química , Quelantes/química , Quelantes de Ferro/química , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Quelantes/farmacologia , Eletroquímica , Fibroblastos/efeitos dos fármacos , Humanos , Quelantes de Ferro/farmacologia , Ácidos Isonicotínicos/síntese química , Espectroscopia de Ressonância Magnética , Espectrofotometria Ultravioleta , Termodinâmica , Testes de ToxicidadeRESUMO
The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.
Assuntos
Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismo , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Biomassa , Regulação da Expressão Gênica de Plantas , Ácidos Isonicotínicos/farmacologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/genéticaRESUMO
Gamma-aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A )receptor subunits, but also bind the GABA agonist [(3)H]muscimol with a binding affinity in the range reported for other endocrine cells (K(d) = 2.740 +/- 0.721 nM). However, they exhibit a low B(max) value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl(-) currents, changes in resting membrane potential, intracellular Ca(2+) or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an atypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated.
Assuntos
Células Intersticiais do Testículo/fisiologia , Receptores de GABA/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Sinalização do Cálcio/fisiologia , Linhagem Celular , Canais de Cloreto/fisiologia , AMP Cíclico/fisiologia , DNA Complementar/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Agonistas GABAérgicos/metabolismo , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Ácidos Isonicotínicos/farmacologia , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Muscimol/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Patch-Clamp , RNA/biossíntese , RNA/genética , Receptores de GABA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Fluorescência , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Midbrain dopaminergic (DA) neurons in vivo exhibit two major firing patterns: single-spike firing and burst firing. The firing pattern expressed is dependent on both the intrinsic properties of the neurons and their excitatory and inhibitory synaptic inputs. Experimental data suggest that the activation of N-methyl-D-aspartate (NMDA) and GABAA receptors is a crucial contributor to the initiation and suppression of burst firing, respectively, and that blocking Ca(2+)-activated potassium SK channels can facilitate burst firing. A multi-compartmental model of a DA neuron with a branching structure was developed and calibrated based on in vitro experimental data to explore the effects of different levels of activation of NMDA and GABAA receptors as well as the modulation of the SK current on the firing activity. The simulated tonic activation of GABAA receptors was calibrated by taking into account the difference in the electrotonic properties in vivo versus in vitro. Although NMDA-evoked currents are required for burst generation in the model, currents evoked by GABAA-receptor activation can also regulate the firing pattern. For example, the model predicts that increasing the level of NMDA receptor activation can produce excessive depolarization that prevents burst firing, but a concurrent increase in the activation of GABAA receptors can restore burst firing. Another prediction of the model is that blocking the SK channel current in vivo will facilitate bursting, but not as robustly as blocking the GABAA receptors.
Assuntos
Dopamina/metabolismo , Mesencéfalo/citologia , Neurônios/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Receptores de GABA-A/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Apamina/farmacologia , Bicuculina/farmacologia , Simulação por Computador , Interações Medicamentosas , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Ácidos Isonicotínicos/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mesencéfalo/metabolismo , Mesencéfalo/fisiologia , Modelos Neurológicos , N-Metilaspartato/farmacologia , Picrotoxina/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Potássio Ativados por Cálcio de Condutância BaixaRESUMO
Systemic acquired resistance (SAR) to infection by Botrytis cinerea in the leaves of sunflower (Helianthus annuus L.) plants was induced following cotyledon inoculation with B. cinerea or treatment with abiotic inducers. Salicylic acid (SA), benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA) or EDTA protected sunflower plants against Botrytis infection, that was revealed by a reduction in the number and area of the necrotic lesions in upper leaves after challenge inoculation with the pathogen. SA and BTH were more potent inducers than INA, EDTA or pre-inoculation with the fungus. In addition to resistance to B. cinerea, the upper leaves have also developed resistance to maceration by a mixture of cell wall-degrading enzymes. Calcium nitrate inhibited both the protective effect and the resistance of leaf discs to cell-wall degrading enzymes. All the tested chemicals increased the synthesis and excretion of sunflower phytoalexins--coumarins scopoletin and ayapin and induced the PR-proteins chitinase and 1,3-beta-glucanase, being the inducer effect of each activator correlated with the level of protection against B. cinerea (BTH > SA > INA > EDTA). Thus, SAR induction is mediated by general increase of plant defence responses. This is the first report on SAR in sunflower.
Assuntos
Helianthus/genética , Imunidade Inata/efeitos dos fármacos , Doenças das Plantas/genética , Anti-Infecciosos/farmacologia , Botrytis/patogenicidade , Compostos de Cálcio/metabolismo , Compostos de Cálcio/farmacologia , Parede Celular/efeitos dos fármacos , Quitinases/biossíntese , Quitinases/efeitos dos fármacos , Ácido Edético/metabolismo , Ácido Edético/farmacologia , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucana 1,3-beta-Glucosidase , Helianthus/efeitos dos fármacos , Helianthus/enzimologia , Helianthus/microbiologia , Ácidos Isonicotínicos/farmacologia , Nitratos/metabolismo , Nitratos/farmacologia , Doenças das Plantas/microbiologia , Extratos Vegetais/biossíntese , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Sesquiterpenos , Terpenos , Tiadiazóis/farmacologia , beta-Glucosidase/biossíntese , beta-Glucosidase/efeitos dos fármacos , FitoalexinasRESUMO
To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.
Assuntos
Arabidopsis/genética , Proteínas de Membrana , Oomicetos/crescimento & desenvolvimento , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Sequência de Aminoácidos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutationa Transferase/metabolismo , Ácidos Isonicotínicos/farmacologia , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Oomicetos/patogenicidade , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ativação Transcricional , VirulênciaRESUMO
A full-length lipoxygenase cDNA (RCI-1) has been cloned from rice (Oryza sativa) whose corresponding transcripts accumulate in response to treatment of the plants with chemical inducers of acquired resistance such as benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA), and probenazole. In contrast, RCI-1 transcript levels did not increase after inoculation with compatible and incompatible races of the rice blast fungus Magnaporthe grisea and the nonhost pathogen Pseudomonas syringae pv. syringae. RCI-1 transcript levels also increased after exogenous application of jasmonic acid, but not upon wounding. Dose-response and time course experiments revealed a similar pattern of transcript accumulation and lipoxygenase activity in BTH-treated rice leaves. Enzymatic analysis of recombinant RCI-1 protein produced in Escherichia coli revealed that 13-hydroperoxy-octadecanoic acids were the predominant reaction products when either linoleic or linolenic acid used as a substrate. The RCI-1 sequence features a putative chloroplast targeting sequence at its N-terminus. Indeed, a protein consisting of the putative chloroplast transit peptide fused to green fluorescent protein was exclusively localized in chloroplasts, indicating that RCI-1 is a chloroplastic enzyme.
Assuntos
Cloroplastos/enzimologia , Ácidos Isonicotínicos/farmacologia , Lipoxigenase/genética , Oryza/enzimologia , Tiadiazóis/farmacologia , Tiazóis/farmacologia , Sequência de Aminoácidos , DNA Complementar/genética , DNA de Plantas/genética , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Imunidade Inata , Ácido Linoleico/metabolismo , Lipoxigenase/biossíntese , Lipoxigenase/isolamento & purificação , Magnaporthe/fisiologia , Dados de Sequência Molecular , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Ácido alfa-Linolênico/metabolismoRESUMO
Treatment of broad bean leaves with salicylic acid (SA) or 2, 6-dichloro-isonicotinic acid (DCINA) induces resistance against the rust fungus Uromyces fabae resulting in reduced rust pustule density. Light-microscopy studies showed that in induced resistant plants the rust fungus is inhibited immediately after penetration through the stomatal pore. The differentiation of infection structures growing within the intercellular space of the leaf, i.e. infection hyphae and haustorial mother cells, is inhibited. Furthermore, low-temperature scanning electron microscopy studies of freeze fractures revealed protrusions at the tips of infection hyphae growing in induced resistant broad bean leaves. Treatment of in vitro-differentiating rust infection structures with intercellular fluids (IFs) from induced resistant plants confirmed that the fungus is sensitive towards an apoplastic anti-fungal activity only after having formed appressoria. Other legume rusts such as U. vignae and U. appendiculatus were likewise inhibited in the presence of IF from SA-treated broad bean leaves. Heterologous antibodies were used to study changes in the extracellular pathogenesis-related (PR) protein pattern after resistance induction. Western blots indicated that chitinases and beta-1,3-glucanases were present in both induced and control plants. In contrast, PR-1 proteins were newly synthesized in response to SA or DCINA application. The major induced PR-1 protein was purified and exhibited strong differentiation-inhibiting activity towards U. fabae infection structures. We conclude that the inhibition of rust infection hyphae in acquired resistant broad bean plants is mainly due to the anti-fungal activity of this induced basic PR-1 protein.
Assuntos
Antifúngicos/farmacologia , Basidiomycota/patogenicidade , Fabaceae/microbiologia , Folhas de Planta/microbiologia , Proteínas de Plantas/farmacologia , Plantas Medicinais , Basidiomycota/efeitos dos fármacos , Basidiomycota/ultraestrutura , Diferenciação Celular/efeitos dos fármacos , Fabaceae/ultraestrutura , Ácidos Isonicotínicos/farmacologia , Folhas de Planta/ultraestrutura , Proteínas de Plantas/isolamento & purificação , Ácido Salicílico/farmacologiaRESUMO
The spinal cord contains endogenous substances (such as cholecystokinin, FMRFamide, etc.) that can block the analgesic effects of opiates. Anti-opiate actions have been most commonly studied by exogenous administration of receptor agonists and receptor antagonists of these substances. However, we have recently demonstrated that anti-analgesia can be brought under environmental control through Pavlovian conditioning. Whereas analgesia can be conditioned to signals for danger, anti-analgesia can be conditioned to signals for safety. Using this paradigm, we have previously demonstrated that conditioned anti-analgesia can reverse a variety of opiate analgesic states, including those produced by conditioned danger signals, systemic morphine, and intrathecal mu- and delta-opiate receptor agonists. These data raise the question of the generality of anti-analgesia actions. The present series of experiments examined the ability of conditioned anti-analgesia to affect non-opiate analgesic states induced by spinal delivery of GABA(A), GABA(B), 5HT2 + 5HT1, and 5HT3 receptor agonists. While conditioned anti-analgesia had no effect on GABA(A) or 5HT2 + 5HT1 non-opiate analgesias, conditioned anti-analgesia completely blocked GABA(B) and 5HT3 non-opiate analgesias. These findings clearly demonstrate that conditioned anti-analgesia can powerfully modulate non-opiate as well as opiate analgesias and bring into question whether putative anti-opiate neuroactive substances may have broader actions than previously suggested.
Assuntos
Analgésicos não Narcóticos/farmacologia , Condicionamento Clássico , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Medula Espinal/efeitos dos fármacos , Analgesia Epidural , Análise de Variância , Animais , Baclofeno/farmacologia , Biguanidas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Agonistas GABAérgicos/farmacologia , Ácidos Isonicotínicos/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
The roles of salicylic acid (SA) and H2O2 in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a-GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae, SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H2O2 and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H2O2 leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 microM. Leaf disks preincubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H2O2-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.
Assuntos
Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/biossíntese , Pseudomonas/patogenicidade , Salicilatos/metabolismo , Transdução de Sinais , Amitrol (Herbicida)/farmacologia , Sequência de Bases , Transporte Biológico , Catalase/antagonistas & inibidores , Catecóis/metabolismo , Genes Reporter , Glucuronidase/biossíntese , Glucuronidase/genética , Peróxido de Hidrogênio/farmacologia , Ácidos Isonicotínicos/farmacologia , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Plantas Tóxicas , Salicilatos/farmacologia , Ácido Salicílico , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
The purpose of the present study was to examine the effects of gamma-aminobutyric acid (GABA)A and GABAB receptor blockade and activation on the activity of tuberoinfundibular dopaminergic (TIDA) neurons in male rats. The activity of TIDA neurons was estimated by measuring the concentration of the primary dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence. Administration of the GABAA receptor antagonist SR 95531 increased DOPAC concentrations in the median eminence, and decreased plasma concentrations of prolactin, in a dose- and time-related manner. Administration of the GABAA receptor agonist isoguvacine had no effect per se on DOPAC concentrations in the median eminence, but produced a delayed decrease in plasma prolactin concentrations. Isoguvacine pre-treatment prevented the increase in DOPAC concentrations in the median eminence produced by SR 95531. In contrast, administration of the GABAB receptor agonist baclofen decreased DOPAC concentrations in the median eminence, and increased plasma prolactin concentrations in a dose-dependent manner. Administration of the GABAB receptor antagonist 2-hydroxysaclofen had no effect on TIDA neurons per se, but blocked baclofen-induced decreases in DOPAC concentrations in the median eminence and increases in plasma prolactin concentrations. These results indicate that while activation of GABAB receptors inhibits TIDA neurons, these neurons are tonically inhibited by endogenous GABA acting at GABAA but not GABAB receptors.
Assuntos
Dopamina/fisiologia , Hipotálamo/fisiologia , Neurônios/fisiologia , Ácido gama-Aminobutírico/fisiologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipotálamo/citologia , Ácidos Isonicotínicos/farmacologia , Masculino , Concentração Osmolar , Prolactina/sangue , Piridazinas/farmacologia , Ratos , Ratos Endogâmicos , Convulsões/induzido quimicamenteRESUMO
Different derivatives of isonicotinic acid are used widely enough as antimicrobial and antituberculous agents. However, their neurotropic and cardiotropic effects have been studied little. The paper is concerned with investigations of these types of the activity of the new derivatives of isonicotinic acid: beta-phenyl-beta-alanine, l-proline, DL-valine, beta-alanine and DL-threonine synthesized for the first time at the Institute of Fine Organic Chemistry, Academy of Sciences of Armenia.